The human brain contains from 10 to 100 billion neurons, and each has hundreds of connections with neighboring neurons. Making sense of these intricate connections is essential to understanding brain function. Traditionally, electrodes have been inserted into the brain or patched on a neuron to record neuronal cell activities. Electrical signals offer high temporal resolution, but cannot provide information for a large number of cells simultaneously. Additionally, the surgical implantation of the electrodes compromises the system and requires monitoring to prevent infection.
Imaging technology has provided a means to monitor a large population of cells simultaneously. Technologies such as functional MRI, computed tomography (CT), and positron emission tomography (PET) have potential to image brain activity and body function in a noninvasive manner. But, these imaging technologies do not provide the necessary temporal and spatial resolution that is necessary to understand cellular function.
An alternative method is using optical imaging to image cell activity by recording either the calcium transients or the voltage signals by cells. Traditionally, these methods have used organic dyes that alter their optical properties in response to changes in their environment. For example, the calcium indicator fluo-4 increases its fluorescence in response to increased calcium concentration inside the cell. With these chemical indicators, cellular responses in a large number of cells can be recorded with relatively high temporal and spatial resolution. But, the loading of these dyes into tissue or live animals has been difficult. In general, these dyes must be applied by bulk-loading techniques, such as injection into the target tissues or invasive surgical manipulations. The successful use of these chemical indicators is dependent, in large part, on the successful application of the dyes to the target tissues. The use of chemical indicators is limited further by the lack of cell-type specificity.
Genetically encoded sensors (GES) have provided a means to monitor cell activity with high temporal and spatial resolution in a noninvasive manner and allow long-term studies of neuronal activity and morphology. Several fluorescent protein (FP) based indicators are available that monitor changes in calcium concentration, synaptic transmission, voltage activation, and kinase activity. Among them, the most widely used indicator belongs to the category of calcium sensors. Such calcium sensors include cameleon, Camgaroo, Troponeon, and G-CaMP (G-CaMP 1.3 and G-CaMP 1.6). G-CaMP is an engineered protein that contains a calmodulin domain and a calmodulin binding, myosin light chain, kinase, M13 peptide that flanks the circular permutated EGFP (enhanced green fluorescent protein). The binding of calcium to G-CaMP induces conformational changes of the protein and significantly increases its fluorescence when excited at 488 nm. An improved variant of G-CaMP is G-CaMP2. At physiological temperatures, G-CaMP2 fluoresces approximately 22× brighter than G-CaMP and 6× brighter than G-CaMP1.6 (Tallini, Y. N., et al., 2006). G-CaMP2 also exhibits a 4- to 5-fold increase in signal between basal calcium and saturating calcium conditions. While genetically encoded calcium sensors hold great promise for studying calcium signaling in complex organ function, they have not been effectively used in mammals in vivo because of poor intrinsic signal strength, inadequate temperature stability, or perturbing interactions between the sensing molecule and endogenous cellular proteins. Thus, there remains a need for compositions and methods for detecting and measuring, e.g., intracellular signaling in vivo.